Lung cancer is the leading cause of cancer related mortality in both men and women and remains a major health issue. More than 160,000 individuals will die from lung cancer in the coming year, more than breast, prostate and colon cancer combined. The majority of lung cancer cases is attributable to tobacco smoking and in some cases other environmental risk factors. Although the relative risk of developing lung cancer declines dramatically in smokers who quit, former smokers remain at risk for the disease. Several recent studies show that greater than 50% of newly diagnosed lung cancers occur in former smokers. Of the tumors detected in former smokers, nearly 50% occurred in patients who had quit for more than five years. It is estimated that there are approximately equal numbers of smokers and former smokers in the United States. Since smoking cessation is a major public health initiative, former smokers will increasingly account for a higher percentage of lung cancer cases. Thus, two high-risk population groups exist for lung cancer and improved disease management can be beneficial to both current and former smokers. Additionally the prognosis for lung cancer patients is very poor, as reflected by an overall, 5-year survival rate of only 14%. The poor prognosis for lung cancer patients is due, in part, to the historical lack of effective early detection measures. TUMOR SUPPRESSOR GENES ON CHROMOSOME 9P:Chromosome 9p deletions and alterations occur early and often in lung cancer. The p16/CDKN2 locus, located on 9p, is suspected to be the major tumor suppressor gene inactivated in this tumor type. However, we have previously identified a region of homozygous deletion on the short arm of chromosome 9p at the microsatellite marker D9S126. This region is distinct from the p16/CDKN2 tumor suppressor gene (TSG) locus and lies approximately 2 cM proximal. We proposed that the region harbors a TSG that may be important in lung tumorigenesis. To this end we have employed several techniques to identify genes within this chromosomal region. Currently, we have isolated several expressed sequence tags (ESTs) and genes mapping in or around D9S126. Additionally, we have used immunohistochemistry to analyze the expression of p16 in adenocarcinomas and squamous cell carcinomas of the lung and subsets of these tumors were analyzed for loss of heterozygosity (LOH) with microsatellite markers spanning the short arm of chromosome 9. The immunohistochemistry revealed a significant difference in the percent of tumors positive for p16 with the adenocarcinomas having a higher percentage of positive staining than the squamous cell carcinomas. Loss of heterozygosity analysis demonstrated that the pattern of loss was similar between p16 positive and negative squamous cell carcinomas. However, there were moderately significant differences in the LOH analysis between p16 positive adenocarcinomas and squamous cell carcinomas at some of the markers. In a more recent study, 30 non-small cell lung cancer and 12 small cell lung cancer cell lines were screened with 55 markers to identify new regions of homozygous deletion on chromosome 9p. Three novel non-contiguous homozygously deleted regions were detected and ranged in size from 840 Kb to 7.4 Mb. One gene identified in the deletion at D9S126, TUSC1, is an intronless gene. Multiplex PCR and Southern blot confirmed the homozygous deletion of TUSC1. Northern blot analysis of TUSC1 demonstrated two transcripts of approximately 2 and 1.5 kb that are likely generated by alternative polyadenylation signals. Both transcripts are expressed in several human tissues and share an open reading frame encoding a peptide of 209 amino acids. Analyzing cell line cDNAs by RT-PCR demonstrated down regulation of TUSC1 in cell lines with or without homozygous deletions suggesting TUSC1 may play a role in lung tumorigenesis. Taken together, these data suggest that chromosome 9p may contain other tumor suppressor genes important in lung tumorigenesis and that the different patterns of LOH between squamous cell carcinomas and adenocarcinomas of the lung may indicate their locations.THE COT/MAP3K8 GENE IN LUNG TUMORIGENESIS:The Cot/MAP3K8 gene is a MAP kinase kinase kinase expressed in a variety of cells including spleen, thymus, liver, and lung and was originally identified by transfection of a human thyroid cancer DNA into SHOK cells. The gene was found to be oncogenic and constitutively activate when altered at the 3' end. MAP3K8 induces cellular transformation and tumorigenesis by transcriptional over-expression and mutation. However, mutation of the gene appears to be a rare event in humans, but altered Cot/MAP3K8 expression is associated with multiple tumor types. Cot/MAP3K8 possesses the unique characteristic of activating multiple cascades, including both proliferative and apoptotic signal transduction pathways such as the MEK-1 and SEK-1 pathways, respectively. In NIH3T3 transfection assays utilizing lung tumor DNA, our lab identified a 3' alteration of Cot/MAP3K8 similar to the previous reports. We therefore proposed that oncogenic activation of Cot/MAP3K8 might play a role in lung tumor formation and development. The complete open reading frame of Cot/MAP3K8 was examined by polymerase chain reaction, single strand conformational polymorphism (PCR-SSCP) analysis in 40 lung tumor cell lines to identify mutations within the sequence. One mutation/polymorphism was identified in a cell line but did not result in an amino acid change. No other point mutations were found which might activate the gene. Using 3' RACE and DNA sequencing to analyze lung tumor cell lines that might harbor truncations or alterations at the 3' end of Cot/MAP3K8 did not identify any cell lines with activations of the gene through this mechanism. We investigated MAP3K8 expression in lung cancer cell lines to determine if transcriptional aberrations are associated with lung cancer. Primer extension, 5'RACE, and PCR-SSCP characterized the MAP3K8 promoter; Realtime PCR and Western blot analyzed transcriptional and translational expression; and FACS analysis examined effects of MAP3K8 on cell cycling in non-transformed lung cells. We reported that the promoter is a GC rich, TATA-less sequence containing at least six transcription initiation sites. Realtime PCR demonstrated 7/17 non-small cell lung cancer (NSCLC) cell lines significantly increased MAP3K8 mRNA expression, with 3 cell lines over 50-fold greater that of normal lung cells. While 4/14 small cell lung cancer (SCLC) cell lines increased expression, the majority of SCLC cell lines decreased expression. Western blot analysis demonstrated that mRNA and protein expression levels did not correlate in the tumor cell lines but did correlate in transfected non-transformed lung cells. Unexpectedly, stable transfection of either a wild type or mutant MAP3K8 into an immortalized lung cell line slowed proliferation in a non-apoptotic manner, but only the mutant gene arrested cells during G0/G1. These data suggest MAP3K8 expression is altered in lung cancer cells lines with a discordant relationship existing between mRNA and protein levels; whereas non-transformed lung cells maintain a correlation between increased mRNA and protein expression and demonstrate altered cell cycle regulation from MAP3K8 activity.GENETIC SUSCEPTIBILITY TO LUNG CANCER:A multidisciplinary team of researchers has been working on a project for more than a decade to identify lung cancer susceptibility loci in high-risk lung cancer families. The project began when we identified high-risk lung cancer families and began to collect the families and necessary biospecimens for genotyping and linkage analysis. We conducted a genome-wide linkage analysis of 52 extended pedigrees ascertained through lung cancer probands who had several first degree relatives.